Cytotoxic Bromoindole Derivatives and Terpenes from the Philippine Marine Smenospongia sp. Deniz Tasdemir*,a,b, Timothy S. Bugnia, Gina C. Mangalindanc, Gisela P. Concep- cio´ nc, Mary Kay Harpera and Chris M. Irelanda a Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84112, U.S.A. b Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, TR-06100 Ankara, Turkey. Fax: +90-312-3114777. E-mail: [email protected] c Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines * Author for correspondence and reprint requests Z. Naturforsch. 57c, 914Ð922 (2002); received March 21/May 16, 2002 Smenospongia, Bromoindole Derivatives, Pyrroloiminoquinone Alkaloid A detailed chemical analysis of a Philippine marine sponge Smenospongia sp. has been performed. This study yielded four new metabolites, 5-bromo-l-tryptophan (1), 5-bromo- abrine (2), 5,6-dibromoabrine (3) and 5-bromoindole-3-acetic acid (4). The pyrroloiminoqui- none alkaloid, makaluvamine O (5) as well as 5,6-dibromotryptamine (6), aureol (7) and furospinulosin 1 (8) were also isolated. Although 1 and 4 have been synthesized previously, this is the first report on the isolation of these compounds from a natural source. The furan- osesterterpene furospinulosin 1 (8) was obtained for the first time from the genus Smenos- pongia. The structures of all compounds were established by spectroscopic methods (UV, IR, α 1D and 2D NMR, MS, [ ]D). The cytotoxic potential of 1Ð8 was evaluated in a panel of isogenic HCT-116 human colon tumor cell lines.

Introduction Experimental Many brominated indole derivatives have been General experimental procedures isolated from marine sources and shown to exhibit UV spectra were recorded in MeOH on a Hew- interesting biological activities (Davidson, 1993; lett-Packard 8452A diode array spectrophotome- Gribble 1992). In the continuation of our investi- ter. IR spectra were recorded using a Jasco FTIR- gations into the chemistry of marine organisms, we 420 spectrophotometer (NaCl disk). Optical rota- investigated a Smenospongia sp. collected from tions were measured on a Jasco DIP-370 Digital the Philippines. Detailed examination of this Polarimeter (589 nm). NMR spectra were ac- sponge has resulted in the isolation of a variety of quired at 26 ∞C on a Varian 400 MHz instrument. simple indole alkaloids, 5-bromo-l-tryptophan (1), Mass spectra were taken on Finnigan MAT 95 5-bromoabrine (5-bromo-N-methyl-l-tryptophan, (FABMS) and Finnigan LCQ DECA ion trap 2), 5,6-dibromoabrine (5-bromo-N-methyl-l-tryp- (ESIMS) spectrometers. Prediction of EIMS frag- tophan, 3) and 5-bromoindole-3-acetic acid (4). mentation patterns was made by High Chem Mass The pyrroloiminoquinone alkaloid makaluvamine Frontier program (version 2.0). HPLC separations O(5), as well as 5,6-dibromotryptamine (6), were performed on a Varian C-18 semi-prepara- aureol (7) and furospinulosin 1 (8) were also iso- tive column (250 ¥ 10 mm i.d, 4 ml/min) using a lated and characterized. In this paper, we describe Beckman 168 photodiode array system (202 nm). the isolation and the structure elucidation of 1Ð4. For flash chromatography, SiO2 (Merck Kieselgel The cytotoxic activity of all compounds (1Ð8) will 60, particle size 0.040Ð0.063 mm) and C-18 mater- also be presented. ial (J. T. Baker, 40 µm, 275 A˚ ) were used. Sepha- dex LH-20 and d-tryptophan were purchased from Sigma while 5-bromoindole-3-acetic acid was purchased from Aldrich.

0939Ð5075/2002/0900Ð0914 $ 06.00 ” 2002 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com · D D. Tasdemir et al. · Chemical Constituents of the Philippine Sponge, Smenospongia sp. 915

Animal material (MeOH), the residue was dried and identified as 5,6-dibromoabrine (3, 10 mg). The specimen of Smenospongia sp. was col- 5-Bromo-l-tryptophan (1): Brownish amor- lected by SCUBA in Batanes, Philippines, in 1999 α 22 phous powder. [ ]D = Ð24∞ (c = 0.05, MeOH). and kept frozen until the work-up. The sponge was UV (MeOH): 230 (3.87), 290 (3.47). IR (NaCl): identified by one of us (M. K. H.). Voucher speci- 3446, 3408, 3005, 1654, 1577. 1H NMR (400 MHz, mens (# PBat99-3-56) were deposited at both the 13 DMSO-d6) Table I; C NMR (100 MHz, DMSO- University of the Philippines and the University + d6) Table II. HREIMS 207.9769 ([M-C2H4NO2] , of Utah. + C9H7NBr ; calc. 207.9762). EIMS 210 (100, + + [M-C2H4NO2] ), 208 (95, [M-C2H4NO2] ), 195 Extraction and isolation (20), 197 (20), 129 (31), 44 (52). (Ð)-ESIMS 283 [MÐH]Ð, 281 [MÐH]Ð; (+)-ESIMS 285 [M+H]+, + + + Thawed sponge material was soaked in MeOH 283 [M+H] , 268 [M-NH3] , 266 [MÐNH3] . (250 ml) for 24 h and the solution was decanted. 5-Bromoabrine (2): Light brown powder. α 22 α 22 This procedure was repeated three times. The [ ]D = Ð34∞ (c = 0.05, MeOH), [ ]D = + 45.8∞ α 22 combined MeOH extracts (1.59 g) were evaporated (c = 0.05, 1 n HCl), [ ]D = + 46.0∞ (c = 0.05, 1 n to dryness in vacuo to give a brown residue, which NaOH). UV (MeOH): 228 (3.59), 288 (2.92). IR 1 was dissolved in 10% H2O in MeOH (200 ml) and (NaCl): 3234, 2921, 1622, 1394. H NMR 13 partitioned against hexane (3 ¥ 200 ml). The water (400 MHz, DMSO-d6) Table I; C NMR (100 MHz, content of the MeOH phase was then adjusted to DMSO-d6) Table II. HREIMS 209.9731 ([M- + 81 + 30% by adding 80 ml water before partitioning C3H6NO2] ,C9H7 BrN , calc. 209.9741), 207.9750 + 79 + against CHCl3. The hexane extract (291 mg) was ([M-C3H6NO2] ,C9H7 BrN , calc. 207.9762). concentrated and passed through a flash silica col- EIMS 298 (< 1, [M]+), 296 (< 1, [M]+), 208 (100, + + umn using a hexane: EtOAc gradient. The fraction [M-C3H6NO2] ), 210 (100, [M-C3H6NO2] ). eluted with 20% EtOAc in hexanes was purified (+)-ESIMS 299 [M+H]+, 297 [M+H]+. by C-18 HPLC using an isocratic MeCN-H2O 5,6-Dibromoabrine (3): Light brown powder. α 22 α 22 (98:2, v/v) mixture to yield furospinulosin 1 (8, [ ]D = Ð31∞ (c = 0.4, MeOH), [ ]D = + 43.5∞ α 22 8.0 mg). The fraction eluted with 30% EtOAc in (c = 0.05, 1 n HCl), [ ]D = + 44.0∞ (c = 0.05, 1 n hexane was pure aureol (7, 35.0 mg). NaOH). UV (MeOH): 228 (3.69), 290 (2.97). IR 1 The CHCl3 extract (254.0 mg) was fractionated (NaCl): 3448, 3408, 3011, 1660, 1582. H NMR 13 by C-18 FCC using MeOH gradients in H2O. The (400 MHz, DMSO-d6) Table I; C NMR fractions eluted with 30, 50 and 100% of MeOH (100 MHz, DMSO-d6) Table II. HREIMS + 79 + in H2O afforded 5-bromoindole-3-acetic acid (4, 285.8847 ([M-C3H6NO2] ,C9H6 Br2N , calc. 2.2 mg), makaluvamine O (5, 3.0 mg), and 5,6-di- 285.8867). EIMS 290 (3), 288 (6), 286 (3) (all [M- + bromotryptamine (6, 51.8 mg), respectively. C3H6NO2] ). (+)-ESIMS 379, 377, 375 (all + + The aqueous MeOH layer was repeatedly tritu- [M+H] ), 348, 346, 344 (all [M-CH3NH2] ). rated with MeOH to remove insoluble salts before 5-Bromoindole-3-acetic acid (4): Brownish applying to a C-18 flash column. Employment of amorphous powder. 1H NMR (400 MHz, DMSO- 13 a multistep MeOH gradient (0Ð100% MeOH) in d6) Table I; C NMR (100 MHz, DMSO-d6) 79 + H2O gave 11 fractions. The fractions eluted with Table II. HREIMS 252.9757 (C10H8NO2 Br , 40 and 50% MeOH in water were combined. Dur- calc. 252.9738). EIMS 255 (48, [M]+), 253 (48, ing the evaporation of this fraction, a precipitate [M]+), 210 (100), 208 (100), 145 (32), 129 (36), 81 formed, which was identified as 5-bromo-l-trypto- (61), 69 (84). phan (1, 20.0 mg). The MeOH-soluble portion was Makaluvamine O (5). Purple solid. UV (MeOH): purified over Sephadex LH-20 using 70% MeOH 222 (3.81), 244 (3.28), 328 (2.94), 362 (sh, 2.86), in water as eluent. 5-bromoabrine (2) was isolated 542 (2.06). IR (NaCl): 3398, 3002, 2916, 1653. 1H 13 as a light brown powder (2.2 mg). During the NMR (400 MHz, DMSO-d6) Table I; C NMR concentration of the combined C-18 flash CC (100 MHz, DMSO-d6) Table II. HREIMS + 79 + fractions eluted with 60 and 70% MeOH, another 265.9691 ([M] ,C10H7 BrN2O2 , calc. 265.9697); precipitate formed. After filtration and rinsing EIMS 268 (< 1, ([M]+), 266 (< 1, ([M]+), 165 (2), 916 D. Tasdemir et al. · Chemical Constituents of the Philippine Sponge, Smenospongia sp.

103 (3), 91 (100). (+)-ESIMS 269 [M+H]+, 267 PBS. Plates were subsequently incubated for 72 h, [M+H]+. the media aspirated, and 100 µl of fresh medium HCT-116 cell lines. The p53Ð/Ð, p53+/+, p21Ð/Ð, was added. 11 µl of 2.5 mg/ml MTT (3,[4,5-dime- and p21+/+ isogenic cell lines were obtained from thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) Dr. Bert Vogelstein at Johns Hopkins University was added to assess cell viability. Plates were incu- (U.S.A). All cell culture reagents were purchased bated with MTT for 4 hours, the media was aspi- from GIBCO with the exception of the antibiotic/ rated, and 100 µl of DMSO was added to each antimycotic (Sigma). The HCT cells were cultured well. Plates were shaken to dissolve the reduced in McCoy’s 5A medium containing 10% fetal bo- MTT and the absorbance was measured at 570 nm vine serum, 1.0 mm sodium pyruvate, 2.0 mml-glu- using a Multiskan Plus 96-well plate reader. The tamine, 40 units/ml penicillin, 40 µg/ml streptomy- percent survival in each well was determined by cin, and 0.1 µg/ml amphotericin B. Cultures were dividing the absorbance of the test well by the incubated at 37 ∞C in a 5% CO2 atmosphere. Cells average absorbance in wells treated only with were washed with 2 ml EDTA (sodium) in phos- DMSO. All samples were tested in quadruplicate. phate buffered saline (Versene, 1:5000) and har- vested in 3 ml of a 0.25% trypsin 0.03% EDTA Results and Discussion solution. The trypsin was quenched by the addi- The Smenospongia sp. was collected from Ba- tion of 12 ml of culture medium, and the cells pel- tanes (Philippines) in 1999 and stored frozen until leted by centrifugation for five min at room tem- processed. The thawed was extracted with perature. The pellet was resuspended in 15 ml of MeOH. The crude MeOH extract was submitted fresh medium and the cell concentration was de- to our standard solvent-solvent extraction proto- termined using a Coulter counter. Cells were col (see Experimental) and yielded hexane, CHCl3 seeded in 96-well plates (3000 cells/well) in 200 µl and aqueous MeOH-solubles. Each extract was in- of medium and allowed to adhere for 24 h. The dividually chromatographed by a combination of medium was aspirated and replaced with 180 µlof C-18/silica flash column chromatography (FCC), fresh medium. Cells were treated with a 20 µl solu- silica HPLC and Sephadex LH-20 to give 1Ð8 tion of compound dissolved in 10% DMSO in (Fig. 1).

Fig. 1. Bromoindole derivatives (1Ð 6) and terpenes (7, 8) from the Smenospongia sp. D. Tasdemir et al. · Chemical Constituents of the Philippine Sponge, Smenospongia sp. 917

Fig. 2. EIMS fragmentation patterns proposed for bromoindole derivatives, 1Ð4 (α = α cleavage, i = inductive charge transfer)

13 5-bromo-l-tryptophan (1) was isolated as a Table II. C NMR data of 1Ð5 (DMSO-d6, 100 MHz), brownish amorphous powder. Although 1 was pre- δ in ppm viously synthesized (Irie et al., 1984), it was ob- 13C 12345 tained as a natural product in the present investi- 2 125.9 d 125.9 d 127.0 d 125.5 d 124.9 d gation. Compound 1 has been produced as a diet 2a 123.7 s metabolite from corresponding indole derivative 3 109.4 s 109.0 s 109.1 s 108.3 s 19.1 t and serine, catalyzed by Achromobacter petrophi- 4 120.8 d 121.1 d 123.4 d 121.1 d 42.2 t 5 110.9 s 111.0 s 114.8 s 110.9 s lum (Kitajima et al., 1973). The negative and posi- 5a 150.9 s tive ESIMS of 1 gave pseudomolecular ion peaks 6 123.2 d 123.3 d 112.6 s 123.3 d 87.4 s at m/z 281, 283 ([MÐH]Ð); and m/z 283, 285 7 113.3 d 113.2 d 115.9 d 113.3 d 168.3 s ([M+H]+), respectively, in a ratio of 1:1. This was 8 135.0 s 134.9 s 136.0 s 134.8 s 172.1 s 8a 124.5 s indicative of a bromine atom in the molecule. The 8b 117.5 s EIMS of 1 also contained prominent peaks of 9 129.3 s 129.3 s 128.6 s 129.2 s nearly equal intensity at m/z 207.9769 and 210. 10 26.8 t 25.7 t 25.5 t 30.9 t 11 54.7 d 63.3 d 63.2 d 172.5 s The loss of m/z 74 (C2H4NO2) from the even 12 170.0 s 169.0 s 168.9 s [M]+ was compatible with the presence of a 13 (N-Me) 32.3 q 32.2 q NH2CHCOOH terminus (Fig. 2). This data, com- bined with the 13C NMR resonances shown in Table II, fixed the molecular formula as spectrum of 1 revealed three spin-coupled net- δ C11H11BrN2O2. The UV spectrum showed the works. The isolated aromatic proton at 7.26, characteristic indole chromophore with absorp- which is typical of the H-2 of a 3-substituted indole tions at 230 and 290 nm. The 1H NMR spectrum (Thymiak et al., 1985), showed a weak coupling of 1 in DMSO-d6 (Table I) contained four aro- (J = 2.0 Hz) with the NH proton. The other two matic sp2 protons; a diastereotopic methylene, a aromatic proton signals at δ 7.14 and 7.31 consti- methine and a downfield D2O-exchangeable pro- tuted a vicinal pair, which could be assigned as two ton (δ 11.20), which is diagnostic for the NH pro- ortho-positioned protons, H-6 and H-7, respec- ton of the indole ring. The gradient COSY tively, from their homonuclear coupling constants 918 D. Tasdemir et al. · Chemical Constituents of the Philippine Sponge, Smenospongia sp.

1 δ Table I. H NMR data of 1Ð5 (DMSO-d6, 400 MHz). in ppm, J in Hz 1H 1 HMBCs of 12 3 4 5 HtoC 2 7.26 (d, 2.0) 3, 8, 9 7.26 (d, 2.0) 7.31 (s) 7.25 (br s) 7.14 s 3 Ð 2.75 (t, 7.3) 4 7.74 (d, 2.0) 3, 5, 6, 8 7.77 (d, 2.0) 8.02 (s) 7.66 (d, 2.0) 3.60 (t, 7.3) 6 7.14 (dd, 2.0, 8.6) 4, 5, 8 7.15 (dd, 2.0, 8.6) 7.15 (dd, 2.0, 8.6) 7 7.31 (d, 8.6) 5, 9 7.29 (d, 8.6) 7.73 (s) 7.29 (d, 8.6) 10 3.03 (dd, 7.8, 14.8) 2, 3, 9, 11, 12 3.09 (m) 3.14 (dd, 5.1, 12.5) 3.59 s (2H) 3.24 (dd, 4.3, 14.8) 3.15 (dd, 5.5, 14.8) 11 3.50 (dd, 4.3, 7.8) 3, 10, 12 3.35 (dd, 5.5, 11.3) 3.38 (dd, 5.1, 10.9) 13 2.38 s 2.41 s (N-Me) Exch. 11.20 s 2, 3, 8, 9 11.11 s 11.22 s 11.10 s

(JHH = 8.6 Hz). H-6 in turn showed meta coupling 5-Bromoabrine (2) was also isolated as a light with H-4 (δ 7.74 d, J = 2.0 Hz), pointing to a 1,3,4- brown amorphous powder, which showed typical substitution on the benzene ring of the indole. UV and IR absorptions for a tryptophan structure These chemical shifts and the splitting pattern of (see Experimental). In the low resolution ESIMS, the aromatic protons indicated the presence of a 2 showed 1:1 doublet molecular ion peaks [M+H]+ 5-bromo-3-substituted indole fragment (Thymiak at m/z 297 and 299, consistent with the molecular et al., 1985; Djura et al., 1980). This interpretation formula C12H13BrN2O2. The NMR data of 2 were was supported by an HMBC experiment opti- almost identical with those of 1 and were assign- 3 2 mized for 8.0 Hz. All possible JCH and some JCH able to a disubstituted indole moiety, a methylene, correlations were observed (Table I) to favor a nitrogenous methine, a downfield D2O-ex- 5-bromo substition over 6-bromination. The NOE changeable proton and a carboxylic acid (Tables I correlation observed between H-7 and the ex- and II). However, 2 contained an extra singlet at δ 13 δ changeable NH proton further confirmed the posi- H 2.38 that correlated with a C signal at 32.3 tion of the bromine at C-5. The last spin system (q) in the HMQC spectrum. These chemical shift was assigned to the side chain protons, the diaster- values are very typical for an aliphatic N-methyl δ eotopic methylene ( 3.03, dd, J = 7.8, 14.8 Hz, H2- (C-13) function. Hence, the N-methyl substitution 10a; 3.24, dd, J = 4.3, 14.8 Hz, H2Ð10b) and the was predicted to be on the amino group of the side methine proton (δ 3.50, dd, J = 4.3, 7.8 Hz, H-11). chain, and not on the indole nitrogen. The base Analysis of the HMQC spectrum indicated that peaks observed in the HREIMS at m/z 207.9750 the latter proton was attached to a carbon (δ 54.7 and 209.9731 corresponded to the molecular for- α 79 81 d, C-11) to a nitrogen group, probably an NH2. mula of C9H7 BrN and C9H7 BrN, indicating the H-11 showed two bond couplings with both C-10 loss of C3H6NO2 (CHNHCH3COOH, 88 amu) (δ 26.8) and C-12 (δ 170.0). The connection be- from [M]+ as depicted in Fig. 2. HMBC cross tween the side chain (through C-10) and the indole peaks from the N-methyl signal (δ 2.38) to C-10 ring (C-3) ring was readily established by the key (δ 25.7) and C-11 (δ 63.3) unambigiously con- HMBC correlations shown on Table I, thereby firmed the proposed structure for 2. The stereo- completing the planar structure of 1.TheS stereo- chemistry of the only chiral center (C-11) was as- chemistry at C-11 was determined by comparison signed S based on the sign of the specific rotation α 22 α 22 of the specific rotation value of 1 ([ ]D = Ð24.0∞) value ([ ]D = Ð34.0∞, MeOH). Thus, compound α 22 with that of d-tryptophan ([ ]D = +41.6∞, c = 0.5, 2 was identified as 5-bromo-N-methyl-l-trypto- Sigma) in MeOH. The chemical shifts and the phan. A computer search indicated that the non- coupling constants observed for the side chain bromo derivative, N-methyl-l-tryptophan (= ab- protons and carbons further supported that 1 was rine), was previously isolated from several legumi- a α-amino acid derivative. Thus, 1 was identified nous plants (Cannon and Williams, 1982; Kinjo et as 5-bromo-l-tryptophan. al., 1991; Ma et al., 1998). In order to further con- D. Tasdemir et al. · Chemical Constituents of the Philippine Sponge, Smenospongia sp. 919 firm the stereochemistry of C-11, we remeasured synthetic standard (Aldrich). 5-Br-3-IAA has been the specific rotation of 2 in both HCl and NaOH synthesized by Engvild (1977) and we describe its in which abrine was measured (Cannon and Wil- presence in a natural source for the first time. liams, 1982; Kinjo et al., 1991; Ma et al., 1998). The Compound 5 was obtained as a purple solid. λ positive sign observed in each solvent clearly evi- Maxima in the UV spectrum at max 222, 244, 328, denced that 2 had the same (l) stereochemistry as 362 (sh) and 542 nm suggested a pyrroloiminoqui- α 22 1 abrine [(1 n HCl: [ ]D = +45.8∞ for 2, +44.9∞ for none structure (Radisky et al., 1993). The H NMR abrine (Cannon and Williams, 1982); 1 n NaOH: spectrum (DMSO-d6, Table 1) of 5 was decep- α 22 [ ]D = +46.0∞ for 2, +56.1∞ for abrine (Ma et al., tively simple and contained characteristic pyrroloi- 1998)]. minoquinone resonances: an aromatic singlet at δ The positive mode ESIMS of 3 showed a mo- 7.14 (H-2) and two mutually coupled methylene + 79 δ lecular ion ([M+H] ) cluster at m/z 375 ([C12H13 signals at 2.75 (H-3) and 3.60 (H-4), observed as + 79 81 + 13 Br2N2O2] ), 377 ([C12H13 Br BrN2O2] ), 379 triplets with JHH = 7.3 Hz. The C and DEPT-135 81 + ([C12H13 Br2N2O2] ) in a ratio of ca 1:2:1, which NMR spectra of 5 (Table II) displayed ten carbons. is in a good agreement with the presence of two These included two carbonyls whose chemical shifts bromine atoms. The base peaks in the EIMS of 3 are indicative of an o-quinone group (δ 168.3, (m/z 286, 288 and 290, 1:2:1) corresponded to loss 172.1), five quaternary carbons, one aromatic CH + of C3H6NO2 (88 amu) from the [M] , suggesting and two methylenes. The HREIMS of 5 (m/z that 3 possessed the same N-methylated side chain 265.9691 and 267.9) supported the molecular as 2. The NMR data of both the side chain and formula C10H7BrN2O2. The NMR data of 5 in fact the indole substructure of 3 (Tables I and II) were was quite reminescent of makaluvamine-type also very similar to those of 2. The major differ- pyrroloiminoquinones. Spectroscopic comparisons ence originated from the signals of the aromatic between 5 and makaluvone, reported by our re- region due to the addition of the second bromine search group from a Zyzzya sponge (Radisky et al., atom. The two sharp singlets that emerged at δ 1993) suggested both compounds to have a com- 8.02 and 7.73 were assigned to isolated aromatic mon structural motif, except that 5 lacked the 1-N- protons at C-4 and C-7, which could arise if the methyl function (δ 3.82, Radisky et al., 1993) found bromines were at positions C-5 and C-6. The in makaluvone. Hence, the structure of 5 was estab- broad singlet at δ 7.31 that sharpened on exchange lished as 1-N-demethylmakaluvone. After the com-

(D2O) was due to H-2, thus, the side chain was pletion of this manuscript, Hu et al. (2002) have still attached to C-3. The postulated structure was published this compound from the Jamaican Smen- 3 2 confirmed by JCH and JCH HMBC correlations. ospongia aurea, and named makaluvamine O. Of particular help were the pairs H-4/C-8, H-4/ The remaining compounds were identified as C-6, H-7/C-5, H-7/C-9, H-4/C-5 and H-7/C-6. The 5,6-dibromotryptamine (6) (Djura et al., 1980; Van stereochemistry of C-11 was determined on the Lear et al., 1973), aureol (7) (Djura et al., 1980) α 22 basis of the specific rotation data ([ ]D = Ð31∞ and furospinulosin 1 (8) (Cimino et al., 1972) on α 22 α 22 in MeOH, [ ]D = +43.5∞ in 1 n HCl; [ ]D = the basis of comparison of their NMR, MS and α +44.0∞ in 1 n NaOH). Compound 3 is 5,6-dibromo- [ ]D data with those reported previously. N-methyl-l-tryptophan for which we propose the All compounds were screened in HCT-116 colon trivial name 5,6-dibromoabrine. carcinoma cell lines using an MTT assay. Com- Compound 4 was also found to retain the 5- pounds that showed at least moderate cytotoxicity bromo-3-substituted-indole substructure, plus a were further examined in a set of isogenic HCT- degraded side chain composed of an isolated CH2 116 cell lines consisting of p53 and p21 knockouts (δ 3.59 s, 2H) and a carboxylic acid (δ 172.5). From (p53Ð/Ð and p21Ð/Ð) as well as the parental cell the detailed investigation of 1D and 2D NMR line of each (p53+/+ and p21+/+). p53 is one of the spectra (Tables I and II) as well as the HREIMS best studied tumor suppressor genes, which pri- data (m/z 252.9757 and 255), compound 4 was marily functions through its downstream mediator, identified as 5-bromoindole-3-acetic acid (5-Br-3- p21 protein. Since p53 alterations represent the IAA). This assignment was further confirmed by most common mutation found in human tumors, comparison with the 1H and 13C NMR data of the compounds that do not show a p53 dependant 920 D. Tasdemir et al. · Chemical Constituents of the Philippine Sponge, Smenospongia sp.

µ +/+ Ð/Ð +/+ Ð/Ð Table III. IC50 ( m) values of 1Ð8 Compound p53 p53 p21 p21 in isogenic HCT-116 cell lines. 5-Bromo-l-tryptophan (1) > 177 > 177 n.t. n.t. 5-Bromoabrine (2) > 168 > 168 n.t. n.t. 5,6-Dibromoabrine (3) > 133 > 133 n.t. n.t. 5-Bromoindole-3-acetic acid (4) > 197 > 197 n.t. n.t. Makaluvamine O (5) 7179948.6 5,6-Dibromotryptamine (6) 12.6 85 85 63 Aureol (7) 15.9 41 73 61 Furospinulosin 1 (8) 105 141 155 133 Etoposide 3.4 17 3.4 26 n.t.: not tested mechanism would be expected to have increased 1990; Bano et al., 1987). The presence of phytohor- clinical efficacy. Additionally, p21 mutations are mones in has been suggested to be con- also common among human tumors. Identifying nected with a symbiotic relation to the algae compounds with selectivity against genetically de- (Bernart and Gerwick, 1990), although Rasmussen fined cell lines may lead to improved therapies. et al. (1993) have reported several 6-bromoindole Makaluvamine O (5), 5,6-dibromotryptamine derivatives with auxin activity from Pseudosu- (6), aureol (7), and furospinulosin 1 (8) all dis- berites hyalinus, collected at a depth of Ð405 m. played significant activity in HCT-116 cell lines On the other hand, since some bacteria, such as (Table III). With the exception of makaluvamine Pseudomonas, have been shown to produce tryp- O(5), all compounds showed decreased activity tophan derivatives, some with the degraded car- against the p53Ð/Ð cell line indicative of a p53 de- bon chain at C-3 (Narumiya et al., 1979), we can- pendant mechanism. Makaluvamine O (5) showed not exclude the possibility that compounds 1Ð6 a promising activity profile showing very little dif- originate from a symbiotic organism associated ferential between the p53 cell lines while showing with Smenospongia species. The metabolites 1Ð6 µ an order of magnitude lower IC50 (8.6 m) against may also be of dietary origin. Aureol (7), the ses- the p21Ð/Ð cell line. quiterpene hydroquinone, has already been hy- The genus Smenospongia is well known for pro- pothesized to be a dietary metabolite (Thymiak ducing bromotryptamines (Djura et al., 1980; Van et al., 1985). Furospinulosin 1 (8) is a furanosester- Lear et al., 1973) and aureol-type sesquiterpene terpene previously isolated from several sponges phenols (Thymiak et al., 1985; Djura et al., 1980, of the order , such as Hippospongia Bourguet-Kondracki and Guyot, 1989; Bourguet- (Nakamura et al., 1986), Ircinia (Cimino et al., Kondracki et al., 1992). Although derivatives of 1972), Spongia (Erdogan and Sener, 2001) and aplysinopsin, a tryptophan-derived marine natural Fasciospongia (McPail et al., 1998). This is the first product, have also been reported from Smenos- report of 8 from a Smenospongia species. pongia sp. (Thymiak et al., 1985), we believe this Our investigation on the Philippine Smenospon- is the first report of bromotryptophans themselves, gia species has surprisingly afforded a pyrroloimi- from this genus. Abrine, N-methyl-l-tryptophan, noquinone alkaloid, makaluvamine O (5). This has been reported from several terrestrial plants type of compounds are uncommon in nature and (Cannon and Williams, 1982; Kinjo et al., 1991; Ma have thus far been reported from few sponge gen- et al., 1998), but N-methylated tryptophans are era such as Damiria (Stierle and Faulkner, 1991), being described from a marine sponge, again, for Batzella (Sakemi et al., 1989), Prianos (Kobayashi the first time. et al., 1987) and Latrunculia (Ford and Capon, 3-Indole acetic acid (3-IAA) is a well-known 2000). Makaluvamines, specifically, have only been plant growth regulator. Halogenated 3-IAA deriv- obtained from Zyzzya fuliginosa (order Poecilos- atives including 5-bromoindole-3-acetic acid (4, clerida) (Radisky et al., 1993; Schmidt et al., 1995; 5-Br-3-IAA) also show growth promoting activity Venables et al., 1997; Tasdemir et al., 2001; Casa- towards higher plants (Katekar and Geissler, 1983; pullo et al., 2001) and now from two specimens Rasmussen et al., 1995). Marine algae have yielded of a taxonomically unrelated genus, Smenospongia several indole type auxins (Bernart and Gerwick, (order Dicytoceratida). Considering the co-occur- D. Tasdemir et al. · Chemical Constituents of the Philippine Sponge, Smenospongia sp. 921 rence of 5 with 5-bromo-l-tryptophan (1), one can 400 spectrometer was provided by NIH grant speculate that 5 formed by cyclization of 1 fol- RR14768. We are indebted to Dr. Elliot Rachlin lowed by an oxidiation step to elaborate the and Dr. Vajira Nanayakkara for recording mass o-quinonic structure. spectra. We thank Dr. Bert Vogelstein for provid- ing the isogenic HCT-116 cell lines. Special thanks Acknowledgements are due to Pinar Akbay for the assistance at litera- This work was supported by NIH grant CA ture searches. Deniz Tasdemir appreciates the sab- 36622 (C. M. I.). Funding for the Varian Unity batical leave given her by Hacettepe University.

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